Inherited diseases of dental enamel formation are grouped under the designation of amelogenesis imperfecta (AI). About 25% of all amelogenesis imperfecta (AI) cases are caused by the genes encoding four enamel extracellular matrix proteins, AMELX, ENAM, MMP20, and KLK4. Recently we identified mutations in FAM83H that cause AI in six different families suffering from amelogenesis imperfecta. This demonstrates that FAM83H is necessary for proper enamel formation. Virtually nothing is known about the Fam83h protein. Our objective is to learn the roles played by Fam83h in normal and defective enamel formation. Hypotheses: Because the phenotype in people with FAM83H mutations is limited to developing teeth, we hypothesize that Fam83H is expressed during odontogenesis. Because all of the AI- causing FAM83H mutations are dominant nonsense mutations that terminate translation in the last coding exon (exon 5), we further hypothesize that the Fam83h interacts with other cellular proteins to form functional complexes. We propose four Specific Aims: SA 1: To characterize the temporal and spatial pattern of Fam83h expression during odontogenesis and to determine its subcellular localization. SA 2: To isolate Fam83H protein for structural and functional characterization. SA 3: To identify Fam83H interacting proteins. SA 4: To determine if the expression of truncated Fam83h interferes with amelogenesis. Approach: The temporal and spatial pattern of Fam83h expression during odontogenesis is determined by in situ hybridization and immunohistochemistry. Recombinant and native Fam83h are used in structural and functional studies. Fam83h interacting proteins are identified using the yeast two-hybrid system and validated using a mammalian two-hybrid system, co- immunoprecipitation, and Far Western analyses. Normal and mutated Fam83h are expressed in ameloblasts as transgenes and their effects on enamel formation characterized. Heath Relatedness: Patients with inherited enamel defects (AI) have painful, disfigured teeth, lower self-esteem, and perceive themselves as having an inferior quality of life. The discovery that FAM83H is critical for dental enamel formation is a significant advance in our understanding of the etiology of AI. The proposed research may lead to a breakthrough in our understanding of important activities within ameloblasts and the discovery of new candidate gene(s) for AI. PUBLIC HEALTH RELEVANCE: Our group has determined that defects in FAM83H (family with sequence similarity 83 member H) on chromosome 8 cause severe enamel malformations (autosomal dominant hypocalcification amelogenesis imperfecta). Our objectives are to discover the normal function of Fam83h and how defects in this protein result in enamel malformations. Our long term goal is to advance understanding of normal and pathological tooth formation to stimulate improvements in the public's oral health.